Having identified the p38a subproteome, we will next undertake analysis of phosphoproteins within this subproteome using a combination of 1DE/2DE, IMAC and selective ion scanning mass spectrometry methods. Specifically, p38a-associated proteins will be isolated by Flag pull-down, separated by electrophoresis, and digested with trypsin. These tryptic peptides will either be subjected directly to precursor ion/neutral loss scan analysis with mass spectrometry, or will first undergo a phosphopeptide enrichment step using IMAC. Putative phosphoprotein identifications will be verified by western immunoblotting. Detailed procedures for IMAC and precursor/neutral loss ion scanning are listed below. II.D. Collaboration with Project 4 The general approach to characterize the Cdk2 subproteome will proceed via three steps (parallel to the analysis of p38a described above). First we will identify members of the subproteome by immunoprecipitation, electrophoresis and LC/MS/MS. Second, we will determine quantitative changes in this subproteome in the ischemic heart using DIGE and ICAT technologies. Third, we will examine phosphorylated members of this subproteome using IMAC and selective ion scanning methods. Aim 1 of Project 4 will examine proteins associated with Cdk2 in the normal and ischemic myocardium using a combination of immunoprecipitation (IP), 1DE/2DE, and LC/MS/MS. The mitochondrial fraction will be resuspended in sucrose buffer, sonicated briefly, and treated with 0.1% DDM (see Sample Preparation below for specific methodology). The mixture will be centrifuged again to pellet mitochondrial membranes, and released mitochondrial proteins in the supernatant will be used for IP. Myocardial cytosolic or mitochondrial fractions will be pre-cleared with protein A/G beads and Cdk2-associated proteins will be isolated by IP with anti-Cdk2 monoclonal antibodies (Santa Cruz, sc-6248). IgG will be used in place of anti-Cdk2 antibody as a negative control. Cdk2- associated proteins, retained on the beads after washing to remove non-specific interactions, will be eluted from the beads and separated by 1DE. Proteins will be excised from the gel, trypsinized and subjected to LC/MS/MS analysis. [Note: The members of Project 4 have worked closely with the Proteomic Core to optimize the purification of Cdk2 complexes by IP and the running of 1DE/2DE gels and will continue to do so for the duration of these studies.] Together, these techniques will provide the first map of the Cdk2 subproteome in the heart. Detailed procedures for 1DE and LC/MS/MS are described below. See Figure 2 for a general overview of the experimental strategy for Projects 3 and 4. Like p38a, Cdk2 is a soluble kinase, and thus we may be able to gain significant information from 2DE separation of Cdk2 complexes, especially those isolated from the cytosol. Also analogous to p38a, we will implement DIGE and ICAT technologies for the quantitation of protein abundance changes within the Cdk2 subproteome. We will also utilize IMAC and selective ion scanning to determine phosphorytation events within the Cdk2 subproteome and to track changes in these modifications in the ischemic myocardium. Extensive methodology for these techniques is provided below. PHS 398 (Rev. 05/01) Page 321 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. CONTINUATION PAGE Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Vondriska, ProteomJC Core) Note: Although the initial analyses of the p38a and Cdk2 subproteomes will employ denaturing techniques on the front end (1DE and 2DE), we are committed to comprehensive characterization of these subproteomes using nondenaturing approaches (such as BN PAGE) as described in Project 1 with VDAC. After the initial characterization of the p38a and Cdk2 subproteomes in years 1and 2 of the PPG, we will analyze native complexes formed by these molecules with a more directed mass spectrometry and immunoblotting approaches in years 3-5 of the PPG. III. VALIDATION STEPS III.A. Quantitative Validation of Immunoprecipitations and Affinity Pull-Downs and Necessary Controls We will perform western immunoblotting experiments to ensure that the pull-downs to isolate the subproteomes of all molecules are quantitatively consistent. Specifically, we will immunoblot for the target of isolation to ensure that the same amount of protein is used under all conditions to isolate protein complexes. For example, in Project 3, we will western blot for p38a on equal-volume amounts of all the Flag pull-downs from different isolations to be certain that changes in protein association with p38a cannot be attributed to changes in the amount of p38a isolated (i.e. the amount of Flag-p38a pulled down in each experiment will be constant). Likewise in Project 4, we will immunoblot for Cdk2 following immunoprecipitation of Cdk2 based on the same principle. In this regard, we can be sure that the amount of Cdk2 isolated in different experiments, and between different treatment groups, is equal, and thus changes in the associating proteins cannot be attributed to changes in the amount of target protein isolated. A second issue of considerable importance is the use of essential control groups for isolation of protein complexes by immunoprecipitation or affinity pull-down. In both cases, proteins samples are incubated with beads alone, prior to addition of antibody or recombinant protein, to "preclear" the lysate of non-specific interactions between sample proteins and the beads. In the case of immunoprecipitation, a second control that is always performed is substitution of the IgG for the antibody against the target of isolation (e.g. when Cdk2-associated proteins are isolated in Project 4, we will perform parallel experiments using mouse IgG in place of the Cdk2 antibody). This step is required to exclude proteins that associate with the nonspecific region of the antibody. For affinity pull-down experiments (e.g. isolation of VDAC native complexes in Project 1), the second control in addition to preclearing with beads is the substitution of HIS-null proteins for HIS-VDAC. This step helps exclude from analysis proteins that associate with the HIS epitope tag. III.B. Functional Validation of Subproteome Candidates: Collaboration with Heart Biology Core In addition to interacting with the individual Projects to perform proteomics experiments, the Proteomic Core will also interact extensively with the Heart Biology Core to facilitate verification of candidate members of the subproteomes. The validation process involves a battery of classical biochemistry, cell and organelle biology and histo-pathology approaches to determine the functional role of the individual molecules in the context of the entire subproteome in vivo. It is important to highlight the close interaction between the Heart Biology Core and the Proteomic Core that will persist throughout this PPG. In Project 1, we will identify the PKCe-VDAC and PP2CK-VDAC associated proteins and generate a bioinformatic map of these subproteomes in the context of the MPT pore as a whole. Once we have identified the members of these subproteomes, we will confirm these findings by testing for co-localization of the candidate molecules with VDAC and other core components of the MPT pore (such as ANT) in the heart using confocal microscopy in collaboration with the Heart Biology Core. When available, we will also use inhibitors and/or activators of these molecules to examine the effects on mitochondrial function. In Project 2, we will determine residues on ANT1 and VDAC that are targeted for phosphorylation in the protected myocardium. Once these residues have been identified, we will collaborate with the Heart Biology Core and Project 2 to test the functional role of these residues using reconstitution assays and genetic manipulation. In Projects 3 and 4, we will identify p38a- and Cdk2- associated proteins, respectively, and will incorporate this subproteomic data with the PKCe-VDAC/PP2Cic-VDAC bioinformatic map being generated in Project 1. Because we will also map cytosolic and mitochondrial complexes in Projects 3 and 4, the findings will further flesh out any role for p38a- or Cdk2-associated proteins in the regulation of the mitochondria from an "inside-out" and "outside-in" perspective. As with Project 1, the Proteomic Core will collaborate heavily with the Heart Biology Core in Projects 3 and 4 to confirm the localization of p38a- or Cdk2- associated proteins to the mitochondria, and to examine colocalization of these molecules with the components of PHS 398 (Rev. 05/01) Page 322 Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b. CONTINUATION PAGE Principal Investigator/Program Director (Last, First, Middle): Ping, Peipei (Vondciska, PrOteOmJC Core) the MPT pore (such as VDAC and ANT) using confocal microscopy. When available, we will also use inhibitors and/or activators of these molecules to examine the effects on mitochondrial function. Overall, there is a bidirectional flow of information and collaboration between the two Cores in this Program Project. As the Proteomic Core unveils novel members of a subproteome, the Heart Biology Core will provide the resources to the individual Projects to test the functional role of these candidates in the cardiac cell. Likewise, as the Heart Biology Core and the individual Projects decipher new information about the functional aspects of these subproteomes, the Proteomic Core will investigate novel protein complexes to further expand our understanding of the signal network. In this regard, the interaction between the Proteomic Core and the Heart Biology Core will aid execution of the experiments proposed in Projects 1-4, however, this interaction will also generate novel hypotheses about the cardioprotective signaling network to be tested in the future.